The envelope protein (env) of HIV-1 plays two critical roles in the initial stages of virus infection: it binds virus to the cell surface via receptors, and undergoes structural changes mediating fusion between the viral envelope and a cellular membrane. While env-CD4 interactions have been well characterized, the mechanisms controlling the subsequent membrane fusion reaction are poorly understood. It is clear, for example, that CD4 expressed in nonhuman cells is often not sufficient for virus infection due to a block in entry, and different HIV-1 strains can exhibit marked cellular tropism: some virus strains can preferentially infect T-cell lines, others infect macrophages. It is now clear that coreceptors for env-mediated fusion are required and we identified two G protein-coupled chemokine receptors, CXCR4 and CCR5, that fulfill this role. These discoveries represent the first identifications of retrovirus coreceptors for viral entry and infection, and offers an exciting opportunity to study viral tropism, env-mediated membrane fusion and virus entry at the molecular level. The long-term objective of this proposal is to obtain a fuller understanding of the roles these coreceptors play in fusion on the molecular level. The Specific Aims of this proposal are: 1) Characterize the role CXCR4, CCR5 and other coreceptors play in the membrane fusion activities of different envs; determine if other related proteins can serve as cofactors; 2) Produce polyclonal and monoclonal antibodies to HIV-1 coreceptors; identify the functionally important domains in CXCR4, CCR5, and other coreceptors required for activity; 3) Characterize the molecular interactions between env, CD4 and coreceptors; identify the regions in these proteins required for these interactions; determine if interactions exist between CD4 and coreceptors, and determine the mechanism of restriction in CXCR4 coreceptor activity in macrophages, and 4) Characterize the env-mediated fusion process in conjunction with CD4 and coreceptor mutants using a video fluorescence microscopy technique that monitors receptor-induced conformational changes in env, and define the role and identify of the domains in the coreceptors required for inducing these changes; and determine if conformational changes in coreceptors occur. The proposed experiments to address these aims are: i) cell-cell fusion experiments with a battery of recombinant HIV-1 envs and coreceptors using a reporter gene assay for cell fusion; ii) generate and characterize anticoreceptor antibodies through peptide immunization and recombinant vaccinia inoculations; iii) generate and analyze panels of site-directed coreceptor mutants; iv) perform a variety of fluid phase and solid phase (ELISA) assays with env, CD4, coreceptors, and antibodies for direct binding, blocking, and co- immunoprecipitation analyses; and v) monitor and analyze receptor and coreceptor-induced env structural changes using a variety of biochemical and biophysical assays.